Cryogenic electron tomography reveals novel structures in the apical complex of Plasmodium falciparum

ABSTRACT Intracellular infectious agents, like the malaria parasite, Plasmodium falciparum, face the daunting challenge of how to invade a host cell. This problem may be even harder when the host cell in question is the enucleated red blood cell, which lacks the host machinery co-opted by many pathogens for internalization. Evolution has provided P. falciparum and related single-celled parasites within the phylum Apicomplexa with a collection of organelles at their apical end that mediate invasion. This apical complex includes at least two sets of secretory organelles, micronemes and rhoptries, and several structural features like apical rings and a putative pore through which proteins may be introduced into the host cell during invasion. We perform cryogenic electron tomography (cryo-ET) equipped with Volta Phase Plate on isolated and vitrified merozoites to visualize the apical machinery. Through tomographic reconstruction of cellular compartments, we see new details of known structures like the rhoptry tip interacting directly with a rosette resembling the recently described rhoptry secretory apparatus (RSA), or with an apical vesicle docked beneath the RSA. Subtomogram averaging reveals that the apical rings have a fixed number of repeating units, each of which is similar in overall size and shape to the units in the apical rings of tachyzoites of Toxoplasma gondii. Comparison of these polar rings in Plasmodium and Toxoplasma parasites also reveals them to have a structurally conserved assembly pattern. These results provide new insight into the essential and structurally conserved features of this remarkable machinery used by apicomplexan parasites to invade their respective host cells. IMPORTANCE Malaria is an infectious disease caused by parasites of the genus Plasmodium and is a leading cause of morbidity and mortality globally. Upon infection, Plasmodium parasites invade and replicate in red blood cells, where they are largely protected from the immune system. To enter host cells, the parasites employ a specialized apparatus at their anterior end. In this study, advanced imaging techniques like cryogenic electron tomography (cryo-ET) and Volta Phase Plate enable unprecedented visualization of whole Plasmodium falciparum merozoites, revealing previously unknown structural details of their invasion machinery. Key findings include new insights into the structural conservation of apical rings shared between Plasmodium and its apicomplexan cousin, Toxoplasma. These discoveries shed light on the essential and conserved elements of the invasion machinery used by these pathogens. Moreover, the research provides a foundation for understanding the molecular mechanisms underlying parasite-host interactions, potentially informing strategies for combating diseases caused by apicomplexan parasites.

The plot shows the distance between the outermost two membranes of the apicoplast ("Apicoplast"), the inner and outer membranes of the mitochondrion ("Mitochondrion"), as well as between the limiting membranes of the two organelles ("Apicoplast-mitochondrion").Apicoplast mean ± SD =12.6±3.6 nm, N=8; Mitochondrion mean ± SD =11.6±2.2 nm, N=8; Apicoplast to mitochondrion mean ± SD =13.6±3.8 nm, N=8; There was no significant difference between the groups, by one-way ANOVA.(A) A single repeating unit of Rings 1 and 2 was segmented in green at three different thresholds.Its molecular size is estimated to be ~16.3MDa at thresholds 1σ, 14.8 MDa at 2σ, and 13.6 MDa at 3σ. (B) The spacing between neighboring units (green and pink) is ~17.3 nm on the outer edge of top Ring 1 and ~21.7 nm on the outer edge of bottom Ring 2. (C) The diameter of Rings 1 and 2 based on the averaged map.The inner and outer outlines of Ring 1 are ~125.6nm and 186.8 nm, respectively.The outer diameter of Ring 2 is ~232 nm.
Figure S1.Distinct densities associated with rhoptry peripheries.(A) A tomographic slice showing a rhoptry with arrows pointing to a layer of continuous density reproducibly observed to line much of the organelle's limiting membrane.Scale bar, 100 nm.(B) Top panel is a tomographic slice showing the rhoptries.Scale bar, 100 nm.Bottom panel is zoomed in view of the square showing densities coating the cytosolic face of the membrane at the rhoptry's anterior end (arrowheads).Scale bar, 50 nm.(C) As for (B) except from a different tomogram.The arrowheads in the zoomed in view point to the densities coating the anterior end of the rhoptries.(D) A different slice of the same tomogram in (C) showing a surface view of the rhoptry neck region and the densities coating it (zoomed in view).(E) As for (D) except showing a surface view of the rhoptry bulbs.Note that the small associating densities apparent at the rhoptry necks are not apparent on the bulbs (zoomed in view).Scale bars, 100 nm or 50 nm for the zoomed in view.

Figure S2 .
Figure S2.The apicoplast and mitochondrion maintain an intimate association in the merozoite cell.(A)A tomographic slice of a whole merozoite showing a mitochondrion (M, presumptively designated based on double-membrane structure and position adjacent to the 4-membrane apicoplast) and apicoplast (A).The arrow marks the apical rings.Scale bar, 200 nm.(B) The distance between membranes was measured along a line profile plot of the inverted pixel values in the region where the two organelles are in closest apposition across.The plot shows the distance between the outermost two membranes of the apicoplast ("Apicoplast"), the inner and outer membranes of the mitochondrion ("Mitochondrion"), as well as between the limiting membranes of the two organelles ("Apicoplast-mitochondrion").Apicoplast mean ± SD =12.6±3.6 nm, N=8; Mitochondrion mean ± SD =11.6±2.2 nm, N=8; Apicoplast to mitochondrion mean ± SD =13.6±3.8 nm, N=8; There was no significant difference between the groups, by one-way ANOVA.(C) Left panel-a tomographic slice showing a mitochondrion (M) and apicoplast (A).Right panela zoomed in view of the square in the left panel showing the area with minimum distance between the mitochondrion and the apicoplast with densities in the interface between them (arrowheads).Scale bars, 100 nm and 50 nm for the left and right panels, respectively.(D) As for (C), except from a different tomogram.
Figure S2.The apicoplast and mitochondrion maintain an intimate association in the merozoite cell.(A)A tomographic slice of a whole merozoite showing a mitochondrion (M, presumptively designated based on double-membrane structure and position adjacent to the 4-membrane apicoplast) and apicoplast (A).The arrow marks the apical rings.Scale bar, 200 nm.(B) The distance between membranes was measured along a line profile plot of the inverted pixel values in the region where the two organelles are in closest apposition across.The plot shows the distance between the outermost two membranes of the apicoplast ("Apicoplast"), the inner and outer membranes of the mitochondrion ("Mitochondrion"), as well as between the limiting membranes of the two organelles ("Apicoplast-mitochondrion").Apicoplast mean ± SD =12.6±3.6 nm, N=8; Mitochondrion mean ± SD =11.6±2.2 nm, N=8; Apicoplast to mitochondrion mean ± SD =13.6±3.8 nm, N=8; There was no significant difference between the groups, by one-way ANOVA.(C) Left panel-a tomographic slice showing a mitochondrion (M) and apicoplast (A).Right panela zoomed in view of the square in the left panel showing the area with minimum distance between the mitochondrion and the apicoplast with densities in the interface between them (arrowheads).Scale bars, 100 nm and 50 nm for the left and right panels, respectively.(D) As for (C), except from a different tomogram.

Figure S4 .
Figure S4.Rings 1 and 2 of Plasmodium show prominent 34-fold symmetry.(A) A side view of Plasmodium Rings 1 and 2 of are shown in gold.The segmented cut view of the rings shows the inner and outer densities of Ring 1 in pink and red color, respectively and the inner and outer densities of Ring 2 in blue and green color.The yellow density bridges the outer densities of Rings 1 and 2. (B, C) The segmented cryo-EM map of Rings 1 and 2 for the tomogram in (A) are shown from a top (B) and bottom (C) view revealing 34 repeating units.Color scheme as in (A).

Figure S5 .
Figure S5.Views of Plasmodium Rings 1 and 2 from different cells.The averaged map of Rings 1 and 2 was mapped back to three tomograms (each representing a different cell).The different views of the rings showing twisting and bending in different ways suggests that the rings are flexible and the linkage between ring units is strong enough to withstand these torsional forces.

Figure S6 .
Figure S6.Measurements of Plasmodium Rings 1 and 2.(A) A single repeating unit of Rings 1 and 2 was segmented in green at three different thresholds.Its molecular size is estimated to be ~16.3MDa at thresholds 1σ, 14.8 MDa at 2σ, and 13.6 MDa at 3σ. (B) The spacing between neighboring units (green and pink) is ~17.3 nm on the outer edge of top Ring 1 and ~21.7 nm on the outer edge of bottom Ring 2. (C) The diameter of Rings 1 and 2 based on the averaged map.The inner and outer outlines of Ring 1 are ~125.6nm and 186.8 nm, respectively.The outer diameter of Ring 2 is ~232 nm.